PROJECT SUMMARY During an immune response, cells of the immune system engage in dynamic cell to cell communication. This communication is required to orchestrate effective immunity to pathogens but also drives disease-causing tissue inflammation. Though typically accomplished through soluble and cell surface proteins, communication through the select transfer of nucleic acids has recently been proposed as a novel form of cell to cell communication. Pioneering work in tissue culture systems has shown that secretion of ribonucleic acids (RNAs) can be selective as well as impact the function of recipient cells, including immune cells. However, major gaps in knowledge remain in understanding how RNA communication occurs in vivo during immune responses. The purpose of this grant is to develop approaches to identify the sources and targets of extracellular RNA communication during tissue inflammation and test the molecular function of this RNA transfer on immune responses. The proposed work uses innovative in vivo genetic tracing systems for both RNA and the vesicles that carry them. It employs single vesicle flow cytometry to pinpoint and characterize extracellular vesicles loaded with RNA cargo, and it uses sensor libraries to detect RNA species transferred to recipient immune cells in vivo. It also leverages gene network analysis and single cell RNA sequencing to define the molecular impact of transferred RNA species. This work will use various models of allergic lung inflammation in the mouse, which provide an ideal systems for studying acute and chronic tissue immune responses. Lung lining fluid is an accessible biofluid specimen, and we have preliminary data demonstrating changes in immune cell-associated extracellular RNAs at the site of local allergic immune responses in the lung. This supports a role for extracellular RNA communication specifically in the allergic inflammation that drives asthma pathogenesis and offers the opportunity to leverage basic science advances in RNA communication to improve diagnosis and treatment of patients with this common chronic lung disease. Overall, we expect that our studies will help to establish RNA communication as an active and functional process during tissue inflammation, provide new systems to study RNA transfer, and open new avenues for understanding chronic inflammatory disease pathogenesis.